Genetic engineering of plant crops to produce stacked input traits, such as tolerance to herbicides and insect resistance, or value added products, such as polyhydroxyalkanoates (PHAs), requires the expression of multiple foreign genes. The transitional breeding methodology used to assemble more than one gene within a plant requires repeated cycles of producing and crossing homozygous lines, a process that contributes significantly to the cost and time for generating transgenic plants suitable for field production (Hitz, B. Current Opinion in Plant Biology, 1999, 2, 135-138). This cost could be drastically reduced by the insertion of multiple genes into a plant in one transformation event.
The creation of a single vector containing cassettes of multiple genes, each flanked by a promoter and polyadenylation sequence, allows for a single transformation event but can lead to gene silencing if any of the promoter or polyadenylation sequences are homologous (Matzke, M., Matzke, A. J. M., Scheid, O. M. In Homologous Recombination and Gene Silencing in Plants; Paszkowski, J. Ed. Kluwer Academic Publishers, Netherlands, 1994; pp 271-300). Multiple unique promoters can be employed but coordinating the expression is difficult. Researchers have coordinated the expression of multiple genes from one promoter by engineering ribozyme cleavage sites into multi-gene constructs such that a polycistronic RNA is produced that can subsequently be cleaved into a monocistronic RNA (U.S. Pat. No. 5,519,164). Multiple genes have also been expressed as a polyprotein in which coding regions are joined by protease recognition sites (Dasgupta, S., Collins, G. B., Hunt, A. G. The Plant Journal, 1998, 16, 107-116). A co-expressed protease releases the individual enzymes but often leaves remnants of the protease cleavage site that may affect the activity of the enzymes.
Protein splicing, a process in which an interior region of a precursor protein (an intein) is excised and the flanking regions of the protein (exteins) are ligated to form the mature protein (FIG. 1a), has been observed in numerous proteins from both prokaryotes and eukaryotes (Perler, F. B., Xu, M. Q., Paulus, H. Current Opinion in Chemical Biology 1997, 1, 292-299; Perler, F. B. Nucleic Acids Research 1999, 27, 346-347). The intein unit contains the necessary components needed to catalyze protein splicing and often contains an endonuclease domain that participates in intein mobility (Perler, F. B., Davis, E. O., Dean, G. E., Gimble, F. S., Jack, W. E., Neff, N., Noren, C. J., Thomer, J., Belfort, M. Nucleic Acids Research 1994, 22, 1127-1127). The resulting proteins are linked, however, not expressed as separate proteins.
It is therefore an object of the present invention to provide a method and means for making multi-gene expression constructs especially for expression in plants of multiple, separate proteins.
It is a further object of the present invention to provide a method and means for coordinate expression of genes encoding multiple proteins, or multiple copies of proteins, especially proteins involved in metabolic pathways or pathways to make novel products.